The present invention relates to a method for determining cholesterol in low density lipoprotein in a sample such as blood, serum and plasma.
A conventional method for determining cholesterol in low density lipoprotein has been based on fractionation using ultracentrifugation. This method, however, has drawbacks of requiring a particular device and taking much time for measurement.
A general method for determining cholesterol without ultracentrifugation is a method which measures total cholesterol level in a sample, cholesterol levels in high density lipoprotein, and triglyceride level individually to calculate cholesterol level in low density lipoprotein using known Friedewald equation. However, this method also has a problem of poor reliability in terms of reproducibility and accuracy if the sample contains much triglyceride.
Recently, a further method for determining cholesterol in low density lipoprotein without requiring a triglyceride value has been proposed in Japanese Laid-Open Patent Publication No. Hei 10-38888; the method comprises step (a) and step (b) wherein the step (a) extinguishes cholesterol contained in high density lipoprotein, very low density lipoprotein and chylomicron of a sample and the step (b) determines cholesterol level In low density lipoprotein. However, since this method determines cholesterol level from the intensity of coloring of a dye, when blood is the sample, the procedure of extinguishing cholesterol is susceptible to an effect of blood concentration (=hematocrit value) because the original sample is already colored. This method has another drawback of laborious procedure due to requirement of individual reagents for the step (a) and step (b), and addition of each reagent to a sample at different time.
An object of the present invention is to provide a method for determining cholesterol in low density lipoprotein only by a single Introduction of a sample, from which the above-mentioned drawbacks and disadvantages inherent to the conventional method for determining cholesterol based on the intensity of coloring of a dye, that is, susceptibility to the effect of blood concentration and laborious procedure is excluded.
The present invention for solving the above-mentioned problems relates to a method for determining or quatitating cholesterol In low density lipoprotein comprising the steps of (a) measuring total cholesterol level in a sample containing at least high density lipoprotein, low density lipoprotein, very low density lipoprotein and chylomicron, and (b) measuring cholesterol levels in the high density lipoprotein, very low density lipoprotein and chylomicron in the sample, wherein a cholesterol level in the low density lipoprotein is determined by subtracting a value obtained in the step (b) from a value obtained in the step (a).
In a preferred mode of the present invention, the step (a) and the step (b) are performed at the same time.
In another preferred mode of the present invention, in at least one of the step (a) and the step (b), cholesterol is reacted with a cholesterol specific enzyme and an electron mediator, and then electrochemically oxidizes a resulting reduced electron mediator to determine cholesterol level based on an oxidation current value as obtained.
In still another preferred mode of the present invention, the step (b) may also include a procedure to precipitate the low density lipoprotein. It is particularly desirable to precipitate the low density lipoprotein with the use of an antibody against the low density lipoprotein, heparin hydroxide, acylated heparin hydroxide, glucosaminoglycan sulfate, polysaccharide sulfate, lectin, or polyanion/divalent cation.
The polyanion/divalent cation is preferably polyanethole sulfonate/divalent cation, phosphotungstate/magnesium ion, or dextran sulfate/magnesium ion.
In a further preferred mode of the present invention, the step (b) may include a procedure to adsorb the low density lipoprotein onto a carrier containing a functional group that adsorb the low density lipoprotein. As the functional group, there may be exemplified carboxyl group, a silanol group, a phosphonic acid group and/or sulfonic acid group.
In a still further preferred mode of the present invention, the step (b) determines cholesterol level in the presence of N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, a magnesium ion, a polyalkyl oxide derivative having an HLB value between 13 and 15, and a pH buffer.
The present invention also relates to a sensor applicable to the above-mentioned method for determining cholesterol, the sensor comprising a sample supply unit composed of a sample holder, two reaction wells, and two channels connecting the sample holder with each of the two reaction wells, wherein a connecting position of both of the two channels with the sample holder is at the same height or level in order that excess overflowing sample from the sample holder can outpour into the two channels at the same time.
The present invention enables concurrent determination of total cholesterol level in a sample and cholesterol levels in high density lipoprotein, very low density lipoprotein and chylomicron in the sample, as well as determination of cholesterol level in low density lipoprotein, by a single introduction of the sample.